Biomedical subjects

Gerd Schmitz

Publications and source records attributed to Gerd Schmitz.

86 records · Page 5Linked to original sources

PubMed2002

Double deletions and missense mutations in the first nucleotide-binding fold of the ATP-binding cassette transporter A1 ( ABCA1) gene in Japanese patients with Tangier disease.

Tangier disease (TD) is a rare autosomal recessive disease characterized by plasma high-density lipoprotein deficiency caused by an ATP-binding cassette transporter A1 ( ABCA1) gene mutation. We describe three different mutations in Japanese patients with TD. The first patient was homozygous for double deletions of 1221 bp between intron 12 and 14 and 19.9 kb between intron 16 and 31. The breakpoint sequence analyses suggest that it is a simultaneous event caused by double-loop formation through multiple Alu. The second patient was homozygous for a novel mutation of A3198C in exon 19, resulting in Asn935His. The third patient was homozygous for A3199G of exon 19 that leads to Asn935Ser, which is the same mutation found in German and Spanish families. Both Asn mutations involved Walker A motif of the first nucleotide-binding fold.

ATP Binding Cassette Transporter 1↗

PubMed2002

ATP-binding cassette (ABC) transporters in atherosclerosis.

Macrophages play a central role in the initiation and progression of atherosclerotic lesions. In the nascent lesion, macrophages transform into foam cells through the excessive accumulation of cholesteryl esters. Dysfunctional lipid homeostasis in macrophages and foam cells ultimately results in the breakdown of membrane integrity and cell death. Studies within the past 2 years have implicated a defined subset of multispan transmembrane proteins, the ATP-binding cassette (ABC) transporters, in macrophage lipid homeostasis. The recent finding that ABCA1, beyond its function as a major regulator of plasma high-density lipoprotein metabolism, exerts significant antiatherosclerotic activities has provided the first direct evidence for the role of an ABC transporter in the pathogenesis of atherosclerosis.

ATP Binding Cassette Transporter 1↗

PubMed2002

Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains.

We have investigated whether a raft heterogeneity exists in human monocyte-derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X-100 (Triton) or Lubrol WX (Lubrol) detergent-resistant membranes from cholesterol-loaded monocytes were associated with the following findings: (i) Lubrol-DRM contained most of the cellular cholesterol and at least 75% of Triton-detergent-resistant membranes. (ii) 'Lubrol rafts', defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline-phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton- and Lubrol-detergent-resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI-mediated lipid efflux and CDC42 were partially localized in Lubrol- but not in Triton-detergent-resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline-phospholipids from Lubrol rafts, whereas HDL3 additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL3 reduced the lipid content in Lubrol- as well as in Triton-detergent-resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1-dependent and HDL3-mediated lipid efflux.

ATP Binding Cassette Transporter 1↗

PubMed2002

ABCA1: regulation, trafficking and association with heteromeric proteins.

HDL metabolism is crucial in maintaining cellular cholesterol and phospholipid homeostasis and prevention of atherosclerosis progression. Recent work identified the ATP-binding cassette transporter A1 (ABCA1) as the major regulator of plasma high density lipoprotein (HDL) cholesterol responsible for the removal of excess cholesterol from peripheral cells and tissues. Here we discuss some novel aspects of the ABCA1 network: 1) the cellular pathways involved in cholesterol and phospholipid efflux, 2) regulation of ABCA1, 3) sulfonylurea receptor 1 (SUR1)- or cystic fibrosis transmembrane conductance regulator (CFTR)-like function of ABCA1, 4) interaction of the ABCA1 C-terminus with beta2-syntrophin, 5) ABCA1 modulation of the Rho GTPase Cdc42, 6) localization of ABCA1 in plasma membrane microdomains and intracellular sites, 7) differential effects of prebeta-HDL precursors on ABCA1 mediated alpha-HDL particle formation and 8) ABCA1 in platelets and its relation to phosphatidylserine-flippase activity. A complex regulatory network and additional antiatherogenic features that may depend on the composition of prebeta-HDL precursor particles are believed to coordinate ABCA1 function in reverse cholesterol and phospholipid transport. Distinct prebeta-HDL ligand-specific receptor-clusters are involved that may modulate specific signaling pathways with varying outcomes related to prebeta-HDL particle composition, the cell-type and the cellular response status.

ATP Binding Cassette Transporter 1↗

PubMed2002

Regulation of platelet adhesion by oxidized lipoproteins and oxidized phospholipids.

Activated platelets adhere to the endothelium and release vasoactive mediators which induce vasoconstriction and remodeling of the vessel wall. The influence of native and ex vivo oxidized lipoproteins enriched with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC), the major lipid responsible for the biological activity of minimally oxidized LDL (mm-LDL), on platelet adhesion, membrane receptor expression and aggregation was studied. Influence of native and oxidized lipoproteins (5-100 microg protein/ml); ox-PAPC (0.5-50 microg/ml); ADP (1-10 microM) as well as the specific phosphatase 1 and 2A inhibitor okadaic acid (3-10 microM) on platelet adhesion, receptor expression and aggregation was measured. Platelets adhered to all the classes of lipoproteins immobilized in plastic microtiter wells (native lipoproteins: HDL<LDL<VLDL<oxidized lipoproteins<ox-PAPC-enriched lipoproteins). Flow cytometry revealed that lipoproteins increased CD41 expression. Preincubation of platelets with ox-PAPC alone, significantly up-regulated CD62p and CD41 receptors (higher dose) but potently inhibited anti-CD36 MoAb binding. Okadaic acid increased anti-CD41 and decreased anti-CD36 and anti-CD42b MoAbs binding. Neither ox-PAPC nor okadaic acid induced platelet aggregation. CD36 seems to be the main receptor responsible for binding of oxidized lipoproteins, particularly its ox-PAPC epitope. The effect of okadaic acid on CD36 and CD41 argue for the participation of phosphorylation-dependent reorganization of cellular trafficking and microtubule organization by ox-PAPC.

Adenosine Diphosphate↗

PubMed2002

MK-383 (tirofiban) induces a GPIIb/IIIa receptor conformation which differs from the resting and activated receptor.

The platelet integrin alphaIIb beta3 (GPIIb/IIIa) acts as a receptor for fibrinogen, playing a critical role in platelet aggregation. GPIIb/IIIa antagonists, which block the receptor-ligand interaction, have been accused of causing occasional thrombocytopenia, probably via drug-induced platelet activation or immunogenic neoepitopes. We, therefore, analyzed the effects of the GPIIb/IIIa antagonist MK-383 (tirofiban) on platelet activation and GpIIb/IIIa conformation. At a concentration of 10(-7) mol/l, MK-383 completely inhibited fibrinogen binding to in vitro stimulated platelets. Simultaneously, the GPIIb/IIIa expression density increased, similar to that on activated platelets, but no effect on P-selectin expression or the formation of platelet-leukocyte aggregates could be observed, indicating that MK-383 binding did not induce general platelet activation. The GPIIb/IIIa receptor conformation was further analyzed by fluorescence resonance energy transfer analysis between fluorochrome-labeled antibodies against different GpIIb/IIIa epitopes. As a result, MK-383 induced a receptor conformation that differed from the resting as well as the activated receptor as induced by ADP or TRAP-6. This conformational modulation of GPIIb/IIIa presents an interesting mechanism which may be linked to receptor recruitment without inducing general platelet activation.

Flow Cytometry↗

PubMed2002

CD14 signalling in lipid rafts: new ligands and co-receptors.

PURPOSE OF REVIEW: Lipid rafts on monocytes/macrophages provide a dynamic microenvironment for an integrated lipopolysaccharide receptor (CD14)-dependent clustering of a set of receptors involved in innate immunity and clearance of atherogenic lipoproteins. The purpose of this review is to summarize the recent advances in our understanding of CD14-dependent receptor clustering and its relevance in atherogenesis. RECENT FINDINGS: Upon binding of various ligands, CD14 as a multiligand pattern recognition receptor induces specific coassembly of additional receptors present on circulating monocytes. SUMMARY: The composition of the receptor cluster and thus the associated signalling pathways defines a ligand specific cellular response, linking endogenous and exogenous host defense to a common recognition platform in rafts.

Animals↗

PubMed2002

Screening for osteoporosis in patients with inflammatory bowel disease by using urinary N-telopeptides.

BACKGROUND: Patients with inflammatory bowel disease are at increased risk of osteoporosis. DESIGN AND METHODS: We carried out a prospective study of bone mineral density and biochemical markers of bone metabolism like osteocalcin and urinary N-telopeptides in 72 patients with inflammatory bowel disease and evaluated if one of these markers detects osteoporosis. In addition, bone mineral density and N-telopeptides were analysed retrospectively in a second series of 93 patients with inflammatory bowel disease in order to assess predictive values found in the first patient group in an independent sample. RESULTS: Multiple linear regression showed that N-telopeptides (P < 0.0001) and total white blood cell count (P = 0.006) correlated negatively with the bone mineral density of the lumbar spine and only N-telopeptides (P = 0.005) correlated negatively with the bone mineral density of the femoral neck. Using receiver operator characteristic curves N-telopeptide concentrations of > 40 (30) nmol N-telopeptides/mmol creatinine were chosen as best cut-off values to exclude osteoporosis at the lumbar spine (femoral neck). Using these cut-off values a negative predictive value of 100% (100%) and a positive predictive value of 37.5% (27.9%) were found in the first group, and a negative predictive value of 95.2% (96%) and a positive predictive value of 15.6% (23.3%) in the second, independent group of patients. CONCLUSION: Our data suggest that N-telopeptide levels could be used as a tool for the screening of osteoporosis and for selecting those inflammatory bowel disease patients where bone mineral density measurement is indicated.

Adult↗

PubMed2002

Native LDL upregulation of ATP-binding cassette transporter-1 in human vascular endothelial cells.

ATP-binding cassette transporter-1 (ABCA1) mediates the lipid efflux from cells to apolipoproteins. In studying the gene expression and regulation of ABCA1 in human vascular endothelial cells (ECs), we found that native low density lipoprotein (LDL) elevates ABCA1 in both protein and mRNA levels in a time- and dose-dependent fashion. Transfection of full-length human ABCA1 in ECs lowers cellular cholesterol content and increases apolipoprotein (apo) A-I-mediated cholesterol efflux. Transfection of the ABCA1 promoter-luciferase reporter results in a 2-fold induction after LDL exposure. The responsive element was mapped within -116 to -54 of the promoter region with use of promoter deletion constructs, as reported in other cells. A mutation of the DR4 site greatly diminished the LDL effect. Results showing that LDL increases the liver X receptor responsive element (LXRE)-driven luciferase activity demonstrate the effect of LDL on LXR activation. Furthermore, ligands of the retinoid X receptor and LXR activate ABCA1 in ECs at levels of both promoter activation and mRNA induction. Therefore, ABCA1 is expressed in vascular ECs and is transcriptionally upregulated by LDL. Overexpression of ABCA1 in these cells prevents overloading of cholesterol by increasing the efflux of cholesterol. Thus, ABCA1 plays an important role in the homeostasis of cholesterol in the vascular endothelium.

ATP Binding Cassette Transporter 1↗

PubMed2002

High-speed detection of the two common paraoxonase polymorphisms Leu55-->Met and Gln192-->Arg by real-time fluorescence PCR and melting curves.

Human paraoxonase (PON1) is a calcium-dependent esterase exclusively bound to apolipoprotein A-I and clusterin, containing high-density lipoprotein (HDL) particles that hydrolyzes organophosphates and aryl esters. Several studies have indicated that PON1 can prevent low-density lipoprotein (LDL) oxidation by hydrolyzing lipid peroxides in the lipoprotein, which is the crucial first step for atherogenesis. Therefore it may protect against the development of atherosclerosis. Serum PON1 activity has been shown to be decreased in familiar hypercholesterolemia and in diseases that are associated with accelerated atherogenesis. The PON1 gene has two common coding region polymorphisms, Leu55-->Met and Gln192-->Arg. Both polymorphisms have been identified as independent risk factors for cardiovascular disease in diabetic and non-diabetic patients. We have established high-speed and easy-to-perform genotyping for the two most significant PON1 gene polymorphisms, employing the LightCycler technology and melting curves. This technique eliminates PCR contamination related to sample handling and does not require digestion of PCR products with restriction enzymes and/or fragment separation on gels.

Amino Acid Substitution↗

PubMed2002

Polymorphisms in the lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein in patients with myocardial infarction.

Gram-negative bacterial infection, namely Chlamydia pneumoniae has been recently discussed as a risk factor for myocardial infarction. The lipopolysaccharide-binding protein (LBP) and the bactericidal/permeability-increasing protein (BPI) play a role in the processes leading to recognition and neutralisation of the Chlamydia pneumoniae and their endotoxins lipopolysaccharides (LPS). LPS interact with plasma LBP, and LBP-LPS complex activates monocytes/macrophages, which can influence the atherosclerotic process. BPI is cytotoxic for Gram-negative bacteria and BPI-LPS complexes do not activate monocytes. We have analysed the polymorphisms in the LBP gene (Gly98-->Cys; Pro436-->Leu) and BPI gene (Lys216-->Glu; PstI polymorphism in intron-5; G545-->C) in 313 patients after myocardial infarction (MI) and in 302 control individuals. Genotype frequencies in the LBP gene and BPI gene did not differ between MI patients and control individuals. Our findings suggest that LBP and BPI polymorphisms do not influence the risk of MI.

Acute-Phase Proteins↗

PubMed2002

ATP-binding cassette transporters in macrophages: promising drug targets for treatment of cardiovascular disease.

Numerous ATP-binding cassette (ABC) transporters are expressed in monocyte-derived macrophages and are subject to sterol-dependent regulation. ABCA1 has been identified as a key regulator of macrophage cholesterol efflux and HDL-mediated reverse cholesterol transport. Although the precise mechanisms of ABCA1 function are not completely understood, recent data suggest that the ABCA1 pathway regulates vesicular traffic, filipodia formation and lipid microdomains, thereby controlling susceptibility to atherosclerosis. Nuclear hormone receptors including LXR/RXR and PPAR/RXR heterodimers are recognized as direct or indirect regulators of ABCA1 expression and are discussed as potential targets for pharmacological intervention in cardiovascular disease. Future studies clarifying the processes involved in the ABCA1 pathway at the cellular level are expected to identify new and possibly more specific pharmaceutical targets.

ATP Binding Cassette Transporter 1↗

PubMed2002

High-throughput quantification of lysophosphatidylcholine by electrospray ionization tandem mass spectrometry.

BACKGROUND: Lysophosphatidylcholine (LPC) has been suggested to play a functional role in various diseases, including atherosclerosis, diabetes, and cancer mediated by LPC-specific G-protein-coupled receptors. Initial studies provided evidence for a potential use of LPC as diagnostic maker. However, existing methodologies are of limited value for a systematic evaluation of LPC species concentrations because of complicated, time-consuming procedures. We describe a methodology based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) applicable for high-throughput LPC quantification. METHODS: Crude lipid extracts of EDTA-plasma samples were used for direct flow injection analysis. LPC 13:0 and LPC 19:0 were added as internal standards, and the ESI-MS/MS was operated in the parent-scan mode for m/z 184. Quantification was achieved by standard addition. Data processing was highly automated by use of the mass spectrometer software and self-programmed Excel macros. RESULTS: The calibrators LPC 16:0, LPC 18:0, and LPC 22:0 showed a linear response independent of sample dilution and plasma cholesterol concentration for both internal standards. The within-run imprecision (CV) was 3% for the major and 12% for the minor species, whereas the total imprecision was approximately 12% for the major and 25% for the minor species. The detection limit was <1 micromol/L. CONCLUSION: The developed ESI-MS/MS methodology with an analysis time of 2 min/sample, simple sample preparation, and automated data analysis allows high-throughput quantification of distinct LPC species from plasma samples, which could be a valuable tool for the evaluation of LPC as diagnostic marker.

Calibration↗

PubMed1998

Recent Advances in Molecular Genetics of Cardiovascular Disorders - Implications for Atherosclerosis and Diseases of Cellular Lipid Metabolism.

Two developments in molecular genetics will profoundly influence our understanding and the diagnosis of cardiovascular disorders. First, the identification of genes responsible for monogenic and polygenic traits by analysis of e.g. large pedigrees and affected sib pairs provides invaluable data regarding the role of specific genes in common diseases like arteriosclerosis, hypertension, diabetes, thrombosis/hemostasis and obesity. Besides the insights into the underlying pathophysiology, this knowledge will permit to identify persons at high risk for disease development. These patients can then obtain a targeted intervention. The second development is related to the availability of new analytical tools for molecular biology. New methods such as sequencing by hybridisation (SBH), DNA-array technology or matrix assisted laser desorption/ionisation-time of flight mass spectroscopy (MALDI-TOF) permit sequence analysis of complete genes within hours. Automated PCR-technologies with homogenous amplicon detection formats simplify PCR and permit its use in the routine laboratory setting. Considering cardiovascular diseases there is a number of genes involved in lipid metabolism (apolipoproteins, lipoprotein receptors, lipolytic enzymes), thrombosis/hemostasis (platelet receptors, pro- and anticoagulant proteins, fibrinogen, PAI's), hypertension (angiotensin converting enzyme, angiotensinogen) glucose metabolism (glucose transporters, enzymes) and obesity (hormones, receptors), that are interesting candidates for sophisticated genetic risk assessment. Furthermore, there are also gene candidates involved in processes of early atherogenesis and chronic inflammation such as complement proteins, cell adhesion molecules, and cellular receptors and enzymes. Most of these gene candidates were derived from pathophysiologic knowledge and subsequent epidemiological studies. However, it is foreseeable that in the coming years genes will be identified which were not known so far to be involved in cardiovascular diseases. Genetic studies will be of prime importance in this area, as is exemplified by animal models. In the long term, analysis of these candidate genes before the implementation of therapy will permit a targeted intervention approach towards high risk patients. This will reduce the overall costs of health care without reducing the quality.

Journal Article↗